Bee (Apis mellifera L. 1758) wax restores adipogenesis and lipid accumulation of 3T3‐L1 cells in cancer‐associated cachexia condition

Abstract Cachexia is associated with various diseases, such as heart disease, infectious disease, and cancer. In particular, cancer‐associated cachexia (CAC) accounts for more than 20% of mortality in cancer patients worldwide. Adipose tissue in CAC is characterized by adipocyte atrophy, mainly due to excessively increased lipolysis and impairment of adipogenesis. CAC is well known for the loss of skeletal muscle mass and/or fat mass. CAC induces severe metabolic alterations, including protein, lipid, and carbohydrate metabolism. The objectives of this study were to evaluate the effects of bee wax (Apis mellifera L. 1758) (BW) extract on adipogenesis, lipolysis, and mitochondrial oxygen consumption through white adipocytes, 3T3‐L1. To achieve this study, cancer‐associated cachexia condition was established by incubation of 3T3‐L1 with colon cancer cell line CT26 cultured media. BW extract recovered the reduced adipogenesis under cachectic conditions in CT26 media. Treatment of BW showed increasing lipid accumulation as well as adipogenic gene expression and its target gene during adipogenesis. The administration of BW to adipocytes could decrease lipolysis. Also, BW could significantly downregulated the mitochondrial fatty acid oxidation‐related genes, oxygen consumption rate, and extracellular acidification rate. Our results suggest that BW could improve metabolic disorders such as CAC through the activation of adipogenesis and inhibition of lipolysis in adipocytes, although we need further validation in vivo CAC model to check the effects of BW extract. Therefore, BW extract supplements could be useful as an alternative medicine to reverse energy imbalances.


| INTRODUC TI ON
Cachexia refers to a systemic wasting condition that causes muscle debilitating, accompanied by unintentional weight loss.Cachexia is considered as a late consequence of diseases such as heart disease, infectious disease, and cancer.In the case of cancer, cachexia occurs in over 70% of cancer patients and accounts for more than 20% of mortality in cancer patients worldwide (especially 30-50% of mortality in gastrointestinal cancer patients) (Palesty & Dudrick, 2003).
Cancer-associated cachexia (CAC) is known for body weight loss (10% or more within 6 months), especially adipose tissue and skeletal muscle weight with chronic inflammatory conditions (Lee et al., 2022).
Particularly, adipose tissue atrophy is affected by various metabolic complications.Although CAC is a well-studied multi-organ crosstalk, mechanisms of adipose tissue loss have recently been explored, and many concepts remain to be determined (Sun et al., 2020).Generally, adipose tissue in CAC is characterized by adipocyte atrophy, mainly due to high levels of lipolysis and modification of the extracellular matrix, resulting in impairment of adipogenesis, fibrosis, dysfunction of energy metabolism, and chronic inflammation (Sun et al., 2020;Yang & Mottillo, 2020).Consequently, remodeled adipose tissue is able to secrete free fatty acids and adipokines in addition to inflammatory cytokines.Adipose tissue consists of heterogeneous cell types such as adipocyte precursor cells, including stromal populations, mature adipocytes, immune cells, vascular cells, lymph nodes, blood vessels, and nerve cells.Loss of adipose tissue in CAC has been explained by increased lipolysis due to lipid mobilization and impaired adipogenesis in adipose tissue (Daas et al., 2018).
In general, adipocytes are mainly involved in the storage of excess fatty acids (FAs) in lipid droplets (LDs) in the form of triacylglycerols (TAGs).During energy-demanding status, stored TAGs are breaking into FAs and glycerol.Adipogenesis is the process of the formation of mature adipocytes from undifferentiated preadipocytes (Jung et al., 2023).Also, adipogenesis is a well-known multi-step cascade requiring specific transcriptional factors, including peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding proteins (C/EBPs) such as C/EBPα, C/ EBPβ, and C/EBPδ.Upon induction of adipogenesis, C/EBPβ and C/ EBPδ levels are increased and subsequently induce the expression of PPARγ and C/EBPα (Lee, Cho, et al., 2020;Tang et al., 2004).C/ EBPα and PPARγ are crucial for terminal differentiation into adipocytes through the induction of adipocyte functional molecules, such as adipocyte protein 2 (aP2), lipoprotein lipase (LPL), perilipin, and fatty acid synthase (FAS).Moreover, lipolysis breaks down TAGs through lipase into FAs and glycerol.The major lipases are hormone-sensitive lipase (HSL), patatin-like phospholipase domain containing-2 (PNPLA2)/adipocyte triglyceride lipase (ATGL), and monoacylglycerol lipase (MGL).
In the aspect of adipogenesis under cachexia, several studies have reported that cachexia has been shown to suppress adipogenesis in in vitro co-culture and in vivo models (Batista Jr. et al., 2012;Bing et al., 2006;Lopes et al., 2018).To discover natural products reversing the cachectic effect on adipogenesis, we screened natural products and found that bee (Apis mellifera L. 1758) wax (BW) restores adipogenesis in cancer-associated cachexia condition.
Treatment of BW on adipocytes was limitedly studied compared to bee venom.However, bee venom has been reported to inhibit adipogenesis in 3T3-L1 adipocytes and to reduce adipogenesis in high-fat-dieted mice (Cheon et al., 2017;Kim et al., 2020).However, the effect of BW on adipogenesis and lipolysis activity under cancer cachectic conditions has not yet been investigated.With this background, the objective of this study was to investigate the effects of BW on adipogenesis under cachectic conditions, lipolysis, and mitochondrial oxygen consumption, using white adipocytes 3T3-L1 cells.

| Bee wax extraction
Beehive wax was collected in Yeongwol, Ganwon-do, South Korea.
The collected bee wax was dried and ground to powder.A total of 1 kg of ground bee hive wax powder was extracted by reflux of distilled water at 100°C for 3 h, filtered (53 μm sieve, Sigma-Aldrich, USA), and lyophilized to get the BW extract powder.

| Total RNA extraction and quantitative real-time PCR
From the differentiated 3T3-L1 adipocytes, total RNA was extracted using the TRIzol method (Ambion, Carlsbad, CA, USA), and quantitative real-time PCR (qRT-PCR) was performed (Lee, Kim, et al., 2020).
RNA (1 μg) was converted into cDNA (Thermo Fisher Scientific, Cleveland, OH, USA), and SYBR Green was used to quantify the gene expression (SYBR Green PCR Master mix, Enzynomics, Korea).
qRT-PCR was performed on the QuantStudio 7 Flex system (Thermo Fisher Scientific, Cleveland, OH, USA).The relative gene expression was determined using the comparative CT method (Kim, Kim, et al., 2023), and the mRNA levels were normalized to the housekeeping gene, cyclophilin.The details of the primer sequences are given in Table S1.

| Oil Red O staining
Mature adipocytes were fixed with 4% formaldehyde for 15 min at room temperature (RT), and the cells were rinsed with phosphatebuffered saline (PBS).Then, the cells were stained with 0.3% Oil Red O solution (Sigma-Aldrich, St Louis, MO, USA) at RT for 1 h to assess lipid accumulation (Nakano et al., 2023).Finally, the cells were washed with distilled water and observed under the light microscope for lipid droplet formation (Evos 5000, Invitrogen, USA).Oil Red O staining was quantified at 520 nm after elution with 100% isopropanol (Lee, Jang, et al., 2021).

| Cell viability analysis
To check the effect of BW extract on cell proliferation, a cell counting kit (CCK-8 kit; Sigma-Aldrich, St Louis, MO, USA) was used (Yan et al., 2023).3T3-L1 cells were sown (3 × 10 3 cells/well) in 96-well plates and treated with various concentrations of BW extract (50, 100, 200, and 400 μg/mL), incubated at 37°C for 48 h.Then, cells were incubated with the added CCK-8 reagent for another 2 h.The cell viability was assessed by the absorbance at 450 nm wavelength using SpectraMax iD3 (Molecular Devices, San Jose, CA, USA).

| Lipolysis assay
The effect of BW extract on lipolysis was quantified using a free glycerol reagent (Sigma, USA), as measured by the rate of glycerol release based on the instructions (Lee et al., 2018).Briefly, cells were incubated in DMEM containing 2% FA-free BSA (Sigma-Aldrich, USA) with/without 1 μM isoproterenol (Sigma-Aldrich, USA), a nonspecific β-adrenergic receptor (β-AR) agonist.The released glycerol level in supernatants was determined spectrophotometrically by measuring the absorbance at 540 nm with SpectraMax iD3 microplate reader.Pierce BCA protein assay reagent (Thermo Fisher Scientific, Cleveland, OH, USA) was used to normalize the glycerol amount to the total protein levels.

| Statistical analysis
Data are represented as the mean ± SD.Statistical analysis was performed using GraphPad Prism software version 10.0.0 (GraphPad Software, San Diego, CA, USA).More than two group comparisons were conducted using two-tailed unpaired Student t-tests, the oneway ANOVA or the two-way ANOVA, followed by post-hoc analysis (Lee, Kim, et al., 2021).Statistical significance was defined as *p < .05,**p < .01,and ***p < .001.

| BW extract has a protective effect against cachexia-associated adipogenesis inhibition
To investigate the effect of BW extract on adipogenesis under cachexia, we made a cachexia mimic condition with 5% CCM (Chen et al., 2020;Han et al., 2022).Although 5% CCM completely blocked the adipogenesis induced by adipogenic stimulation, BW extract significantly restored the adipocyte differentiation suppressed by CCM (Figure 1b). 10 μg/mL BW extract showed marginal effects, but 50-200 μg/mL BW extract increased adipocyte differentiation in a dose-dependent manner, which was assessed using Oil Red O staining.Adipogenesis inhibited by 5% CCM was recovered by 12.6%, 24.5%, and 51% at concentrations of 50, 100, and 200 μg/ mL, respectively (Figure 1c).This result implies that BW extract has a protective effect against cachexia-associated adipogenesis inhibition.Based on these protective effects and cytotoxicity data (Figure S1), 50 and 100 μg/mL concentrations of BW extract were chosen for the following mechanistic experiments.

| BW extract enhances adipocyte differentiation and lipid accumulation during the adipogenesis of 3T3-L1 cells
To explain the protective effect of BW extract against cachexia, we first tested the effect of BW extract on adipocyte differentiation under normal adipogenesis-inducing conditions.Adipogenesis is regulated by specific transcriptional factors, PPARγ and C/EBPs, which are key regulators of adipocyte differentiation.C/EBPα and PPARγ are well known for acting as mediators for the initiation of adipocyte differentiation through increasing expression of their target genes, such as FABP4 (aP2) and adiponectin (Farmer, 2006;Rosen et al., 2000;Wang et al., 2013).BW extract significantly increased the expression of adipogenic genes from the early stages of adipocyte differentiation to the late stages of adipogenic development (Figure 2b).BW extract considerably increased the expression of C/EBPα, PPARγ, and its target gene aP2, which is a marker of mature adipocyte differentiation (Figure 2b).Additionally, adipocyte differentiation is accompanied by the activation of lipogenesis-related genes and results in lipid droplet formation (Pantoja et al., 2008).Therefore, the effect of BW extract on lipid droplet formation was tested in differentiated 3T3-L1 cells.
The representative images showed that BW extract dramatically increased lipid accumulation in a dose-dependent manner (Figure 2c).
Adipogenesis was increased by 17.3% and 40.9% at concentrations of 50 and 100 μg/mL, respectively (Figure 2d).These results suggest that BW extract could upregulate lipid accumulation in 3T3-L1 cells during adipogenesis.

| BW extract downregulates the lipolysis in mature 3T3-L1 adipocytes
The main function of adipocytes is to store excess FAs as TAGs in the cytoplasmic organelles called lipid droplets.When energy demand is increased, stored TAGs are broken down into FAs and glycerol, a process known as lipolysis.The canonical pathway of lipolysis in adipocytes is involved in three major lipases, such as ATGL, HSL, and MGL (Yang & Mottillo, 2020).Most studies have reported dysregulation of lipolysis in adipocytes in metabolic disorders such as obesity, diabetes, liver disease, and cancer.Especially, regulation of lipolysis in adipocytes through oriental medicine could be applied to the treatment of CAC in adipose tissues.Therefore, the effects of BW extract on lipolysis and its related genes in differentiated 3T3-L1 adipocytes were tested in the present study.Lipolysis of completely differentiated 3T3-L1 adipocytes was facilitated by isoproterenol as a non-specific β-AR agonist.During adipogenesis, no significant changes were observed in basal lipolysis level with BW extract treatment compared to control.Whereas, BW extract significantly reduced isoproterenol-induced lipolysis by 32.1% (Figure 3a).These results were further confirmed by qRT-PCR; treatment of BW extract with isoproterenol did not show any significant inhibition in the expression of lipolysis-associated genes ATGL and HSL (Figure 3b).
Taken together, the results imply that BW extract could inhibit lipolysis in mature 3T3-L1 adipocytes without making any changes in lipolysis gene expression.

| BW extract inhibits mitochondrial fatty acid oxidation and glycolysis in mature 3T3-L1 adipocytes
Upon upregulation of lipolysis, metabolites such as FAs and glucose could be provided as substrates for mitochondrial activities.
So, the effect of BW extract on mitochondrial oxidation-related gene expression in fully differentiated 3T3-L1 adipocytes was examined.The expression of mitochondrial fatty acid oxidationrelated genes CPT1α and Acox1 was significantly downregulated by 19.9% and 31.4%,respectively, by 100 μg/mL BW extract (Figure 4a).Then, the efficacy of BW extract on mitochondrial OCR in mature adipocytes was evaluated.BW extract at 100 μg/ mL concentration significantly reduced mitochondrial OCR by 9.54% and 11.39% in basal and maximal respiration, respectively (Figure 4b).In addition to mitochondrial fatty acid oxidation, the extracellular acidification rate (ECAR) is a measure of lactic acid levels and indicates the conversion of glucose to lactate during glycolysis.The ECAR level also decreased by 35.4% upon BW extract treatment (100 μg/mL) in 3T3-L1 cells (Figure 4c).Thus, these results suggest that BW extract might reduce mitochondrial activity by reducing lipolysis as well as lactic acid levels during glycolysis.

| DISCUSS ION
Generally, CAC is well-known for the loss of skeletal muscle and/ or fat mass (Kim, Jung, et al., 2023;Ryden & Arner, 2007;Weber et al., 2022).Patients with CAC have severe metabolic alterations, such as protein, lipid, and carbohydrate metabolism.Some studies showed that the CAC-associated metabolic disorder would possibly be reversed by nutritional supplements (Fearon et al., 2011;Somerville & Koornneef, 2002).A few studies have demonstrated the effects of BW extract in adipocytes and high-fat-dieted rats on adipogenesis (Evershed, 1999;Issara et al., 2019Issara et al., , 2020;;Sugita et al., 2013).Although BW extract treatment for 24 h decreased lipid accumulation and lipogenic-related genes in 3T3-L1 mature adipocytes (Issara et al., 2019), BW extract restored adipocyte differentiation and lipid aggregation, which are reduced by CCM (Figure 1).In vitro adipogenesis results showed a significant increase in lipid accumulation and adipogenic-related gene expression during adipogenesis from preadipocytes to mature adipocytes (Figure 2).Moreover, in line with adipogenic capacity, BW extract treatment could downregulate lipolysis genes and mitochondrial OCR in fully differentiated 3T3-L1 adipocytes (Figures 3 and 4).
Based on the results presented in this study, BW extract could be used as a potential supplement in anti-cachexia therapy and might be an alternative medicine to reverse energy imbalance.In the aspect of active ingredients in BW, various flavonoids, such as chrysin, galangin, pinobanksin, pinocembrin, and techtochrysin, were identified in BW (Tomás-Barberán et al., 1993).
Pinobanksin and pinocembrin affect energy homeostasis by activating a free fatty acid receptor 4 involved in adipogenesis regulation (Cho et al., 2020).Galangin and pinocembrin have a combinatorial/synergistic effect on insulin sensitivity by regulating Akt/mTOR signaling (Liu et al., 2018).These compounds are likely to have a major role in the restorative effect of BW extract on cachexia-associated adipose tissue atrophy.However, further characterization of BW extract is needed for the identification of active compounds responsible for increased adipogenesis in CAC conditions.Thus, further research is required to elucidate the detailed mechanism of action of BW extract on adipogenesis restoration under CAC conditions.
A limitation of this study is that it needs further investigation to determine whether the BW extract has the same effects with regard to adipogenesis, lipolysis, and mitochondrial oxygen consumption in the in vivo CAC model and in CAC patients as shown in 3T3-L1 adipocytes.Also, further characterization of BW extract is needed for the identification of active compounds responsible for increased adipogenesis in CAC conditions.

| CON CLUS ION
In this study, BW extract showed considerable improvement in adipogenesis and lipid accumulation under CAC conditions as well as adipogenic gene expression and its target gene during normal adipogenesis.Administration of BW extract to adipocytes also decreased lipolysis.In addition, BW extract significantly downregulated the mitochondrial fatty acid oxidation-related genes, OCR, and ECAR.Although we need further investigation into the effects of BW extract in an in vivo CAC model, our results suggest that BW extract could improve adipose atrophy in CAC through the activation of adipogenesis and inhibition of lipolysis in adipocytes.

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I G U R E 1 Effects of bee wax (BW) extract on adipogenesis under cachectic condition.(a) Schematic diagram of treatment of BW extraction during adipocytes differentiation of 3T3-L1 cells under cachectic condition by 5% CT26 conditioned media (CCM).(b, c) Effects of BW extraction on adipogenesis and lipid accumulation under cachectic condition were assessed using Oil Red O staining.Black scale bar, 300 μm.Data represent the mean ± SD. *p < .05,** p < .01,*** p < .001indicate significant differences between groups.The significance was assessed by either the one-way ANOVA, followed by post-hoc analysis versus control.

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Effects of bee wax (BW) extract on adipocyte differentiation.(a) Schematic diagram of treatment of BW extract during adipocytes differentiation of 3T3-L1 cells.(b) Effects of BW extract on the expression of adipogenic genes.(c, d) Effects of BW extract on lipid accumulation assessed by Oil Red O staining.Black scale bar, 100 μm.Data represent the mean ± SD. *p < .05,**p < .01,***p < .001indicate significant differences between groups.The significance was assessed by either the two-way ANOVA (b) or one-way ANOVA (d), followed by post-hoc analysis.

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I G U R E 3 Effects of bee wax (BW) extract on lipolysis and lipolysis-associated gene expression of 3T3-L1 cells.(a) Effect of BW extract on lipolysis after differentiation of 3T3-L1 cells.(b) Effect of BW extract on lipolysis-related gene expression in mature 3T3-L1 adipocyte cells.The data represent the mean ± SD. ***p < .01indicates significant differences between groups and ns indicates not significant.The significance was assessed by either the two-way ANOVA (a) or the one-way ANOVA (b), followed by post-hoc analysis.

F
Effects of BW extract on mitochondrial OCR and ECAR in 3T3-L1 cells.(a) Effects of BW extract on mitochondrial fatty acid oxidation-related gene expression in fully differentiated 3T3-L1 cells.(b) Oxygen consumption rate (OCR) of mature 3T3-L1 adipocytes with the treatment of BW extract.(c) Extracellular acidification rate (ECAR) of 3T3-L1 cells with the treatment of BW extract.The data represent the mean ± SD. *p < .05 and **p < .01indicate significant differences between groups, and ns indicates not significant.The significance was assessed by the two-tailed unpaired Student t-tests.